Metal-enhanced fluorescence: an emerging tool in biotechnologyKadir Aslan1, Ignacy Gryczynski2, Joanna Malicka2, Evgenia Matveeva2,Joseph R Lakowicz2 and Chris D Geddes1,2Over the past 15 years, fluorescence has become the dominant

detection/sensing technology in medical diagnostics and

biotechnology. Although fluorescence is a highly sensitive

technique, where single molecules can readily be detected,

there is still a drive for reduced detection limits. The detection of

a fluorophore is usually limited by its quantum yield,

autofluorescence of the samples and/or the photostability of

the fluorophores; however, there has been a recent explosion in

the use of metallic nanostructures to favorably modify the

spectral properties of fluorophores and to alleviate some of

these fluorophore photophysical constraints. The use of

fluorophoremetal interactions has been termed radiative

decay engineering, metal-enhanced fluorescence or

surface-enhanced fluorescence.

Addresses1 Institute of Fluorescence, Laboratory for Advanced Medical

Plasmonics and Laboratory for Advanced Fluorescence Spectroscopy,

Medical Biotechnology Center, University of Maryland Biotechnology

Institute, 725 West Lombard Street, Baltimore, MD 21201, USA2 Center for Fluorescence Spectroscopy, Department of Biochemistry

and Molecular Biology, Medical Biotechnology Center, University of

Maryland School of Medicine, 725 West Lombard Street, Baltimore,

MD 21201, USA

Corresponding authors: Lakowicz, Joseph R (lakowicz@cfs.umbi.edu);

Geddes, Chris D (geddes@umbi.umd.edu)Current Opinion in Biotechnology 2005, 16:5562

This review comes from a themed issue on

Analytical biotechnology

Edited by Keith Wood and Dieter Klaubert

Available online 21st January 2005

0958-1669/$ see front matter

# 2005 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.copbio.2005.01.001

AbbreviationsMEF mwww.scetal-enhanced fluorescenceSiF silver island filmIntroductionFluorescence experiments are typically performed insample geometries that are large relative to the size ofthe fluorophores and relative to the absorption and emis-sion wavelengths. In this arrangement the fluorophoresradiate into free space. Most of our knowledge andintuition about fluorescence is derived from the spectralproperties observed in these free-space conditions. Thepresence of nearby metallic surfaces or particles can alterthe free-space condition and can result in dramatic spec-iencedirect.comtral changes. Remarkably, metal surfaces can increase ordecrease the radiative decay rates of fluorophores and canincrease the extent of resonance energy transfer [14].These effects result from interactions of the excited-statefluorophores with free electrons in the metal, the so-called surface plasmon electrons, which in turn producefavorable effects on the fluorophore. The effects ofmetallic surfaces include fluorophore quenching at shortdistances (05 nm), spatial variation of the incident lightfield (015 nm), and changes in the radiative decay rates(020 nm). The use of fluorophoremetal interactionsin biotechnology has primarily been referred to as radia-tive decay engineering or metal-enhanced fluorescence(MEF).

The concept of modifying the radiative decay rate isunfamiliar to the majority of fluorescence spectroscopists.It is therefore informative to consider the novel spectraleffects expected by increasing the radiative rate, G. Thequantum yield (Q0) and lifetime of the fluorophore (t0) inthe free-space condition are given by:

Q0 G=G knr (1)t0 1=G knr (2)where knr is the non-radiative rate. The presence of anearby metal (m) surface increases the radiative rate byaddition of a new rate Gm [1,2,5]. In this case, the quantumyield (Qm) and lifetime of the fluorophore (tm) near themetal surface are given by:

Qm G Gm=G Gm knr (3)tm 1=G Gm knr (4)

These equations result in unusual predictions for a fluor-ophore near to a metallic surface. From Equations (1)(4),it can be seen that as the value of Gm increases, thequantum yield increases while the lifetime decreases.This is contrary to most observations where the fluores-cence lifetime and quantum yield nearly always change inunison [1].

These effects have been predicted theoretically [67] andare analogous to the increases in Raman signals observedfor surface-enhanced Raman scattering, except that theeffects on fluorescence result from through-space inter-actions and do not require molecular contact between thefluorophores and the metal. This was first demonstratedby Cotton [8] and has been reviewed by Lakowicz [1].Current Opinion in Biotechnology 2005, 16:5562

56 Analytical biotechnologyAs briefly mentioned in the abstract, the use of metallicnanostructures to favorably alter the intrinsic photophy-sical characteristics of fluorophores in a controllable man-ner is a fairly new concept, most of the work appearing inthe literature within the past three years [15]. However,the historical origins of MEF can be traced back to thefirst observations of Drexhage in 1974 [9]. Here, thepossibility of altering the radiative decay rate was demon-strated by the measurement of the decay times (lifetimes)of a europium complex positioned at various distancesfrom a planar metal surface (i.e. mirror) [9]. The lifetimeof the complex remained a single exponential, but oscil-lated with distance from the mirror. This effect isexplained by the changes in phase of the reflected fieldfrom the mirror on the fluorophore. A decrease in lifetimeis found when the reflected field is in phase with thefluorophores oscillating dipole (i.e. Equation (4)) and anincrease is seen when the reflected field is out of phasewith the fluorophores dipole.

In the present article we review the work carried out todate in this emerging area of biotechnology and describethe effects of different silver nanostructures, which havebeen prepared by various deposition methods, on theemission intensity and photostability of appropriatelypositioned fluorophores. The systems reviewed typicallyemploy fluorophores that are used in many biologicalassays. The silver nanostructures usually consist ofsubwavelength-size nanoparticles of silver deposited oninert substrates. The proximity to silver nanostructuresresults in a preferential increase in intensity of low-quantum-yield fluorophores; the lifetimes decrease asthe intensities increase. We subsequently discuss theuse of MEF for its multifarious applications in biotech-nology; for example, in immunoassays, enhanced ratio-metric sensing and DNA detection.

Methodologies for the preparation of silvernanoparticlesA moderately extensive physics literature exists on noblemetal nanostructures. For MEF more dramatic effectshave been reported for non-continuous surfaces such assilver island and colloid films [1].

For use in medical and biotechnological applications,such as diagnostic or microfluidic devices, it would beuseful to obtain MEF at desired locations in the measure-ment device (i.e. MEF on demand). In this regard, recentstudies on MEF have reported the use of a relativelyfacile deposition of silver particles onto glass slides (i.e.silver island films, SiFs [2]). The possibility of using silvercolloids [10] and anisotropic particles such as silver nanor-ods [11] has also been investigated. In addition to varyingparticle shapes, different techniques for silver deposition,such as light deposition and electroplating of silver ontoelectrodes and/or glass slides, have also been employed toobtain localized enhancement of fluorescence [12,13].Current Opinion in Biotechnology 2005, 16:5562In a typical SiF preparation, silver islands are depositedonto a glass substrate, in a random fashion, by reducing asilver salt with the sequential addition of sodium hydro-xide, ammonium hydroxide and glucose solutions whilethe glass substrate is immersed [2]. Atomic force micro-scopy studies have revealed that the SiFs are 3080 nm insize with approximately 40% surface mass coverage [2].The deposited SiFs display plasmon absorption maximanear 430 nm.

In contrast to SiFs, the preparation of colloidal suspen-sions of silver yields spherical silver particles of homo-geneous size. The preparation of silver colloids is wellestablished and many reports are available in the litera-ture ([10] and references therein).

Anisotropic silver particles are predicted to enhance theemission of fluorescence owing to the increased localexcitation fields around the edges of the particles [1].Recently, a methodology for depositing silver nanorodswith controlled size and loadings onto glass substrates hasbeen reported [11]. In this method, silver nanorods aregrown directly on glass substrates from small sphericalsilver colloids in the presence of a cationic surfactant.Silver nanorods deposited onto glass substrates displaytwo distinct surface plasmon peaks: transverse and long-itudinal, which typically appear at 420 and 650 nm,respectively. The length of the silver nanorods wasdetermined by atomic force microscopy to be 1001000 nm.

In a typical preparation of light-induced deposition ofsilver on glass slides, the silver-colloid-forming solution isprepared by adding trisodium citrate solution to a warmedsilver nitrate solution in a closed chamber on a glass slide.The solution is then irradiated by a collimated HeCd laser[12]. Fractal-like silver nanostructures have also beendeposited onto glass substrates (Figure 1a) [13]. In thiscase, commercially available silver electrodes are placedin deionized water 10 mm apart and a constant current issupplied across the electrodes for a short period of time bya constant current generator. All of these recent silvernanostructure preparations have resulted in attractivesilvered surfaces for MEF applications. Some of theseapplications are described below.

Applications of metal-enhanced fluorescenceto biotechnologyUltrabright over-labeled proteins: a new class of

probes based on MEF

Proteins covalently labeled with fluorophores are widelyused as reagents, for example, in immunoassays or for theimmunostaining of biological specimens with specificantibodies. In these applications, fluorescein is one ofthe most widely used probes. An unfortunate property offluorescein is self-quenching, which results from Forsterresonance energy transfer between nearby fluoresceinwww.sciencedirect.com

Metal-enhanced fluorescence: an emerging tool in biotechnology Aslan et al. 57

Figure 1

Fluorescence enhancement from silver nanostructures. (a) Silver fractal-like structures grown on silver electrodes [13]. (b) Photograph of

fluorescein-labeled human serum albumin (molar ratio of fluorescein/ human serum albumin = 7) on quartz and on SiFs as observed with

430 nm excitation and a 480 nm long-pass filter. The excitation was progressively moved from the quartz side to the silver side. (Figure adapted

from [15] with permission.)molecules (homotransfer) [14]. As a result, the intensity ofa labeled protein does not increase with increased extentsof labeling, but actually decreases [15].

It has been reported that self-quenching can be largelyeliminated by the close proximity to SiFs [15,16]; theemission intensity of fluoroscein isothiocyanate-labeledhuman serum albumin (FITC-HSA) with a labeling ratio(fluorophore per protein) of seven is approximately 17times larger in the presence of SiFs than the emission onglass alone [15]. It has been speculated that the decreasein self-quenching results from an increase in the rate ofradiative decay, Gm. The difference in the intensity ofheavily labeled human serum albumin on glass and onSiFs is shown pictorially in Figure 1b. The effect isdramatic, as seen from the nearly invisible intensity onquartz (left-hand side of the slides) and the bright imageon the SiFs (right-hand side of the slides) in this unmo-dified photograph. These results suggest the possibilityof ultrabright labeled proteins based on high labelingratios, and the release of self-quenching using MEF[15].

DNA hybridization

The detection of DNA hybridization [17] forms the basisof a wide range of biotechnological and diagnostic appli-cations, such as gene chips [18,19], PCR [20,21] andfluorescence in situ hybridization [22]. In all these appli-cations increased sensitivity is desirable, particularly forthe detection of a small number of copies of biohazardagents. Also, it would be valuable to have a generalapproach to detect changes in fluorescence intensity uponhybridization. In general, the detectability of a fluoro-phore is determined by two factors: the extent of back-www.sciencedirect.comground emission from the sample and the photostabilityof the fluorophore. A highly photostable fluorophorecan undergo about 106 excitationrelaxation cyclesbefore photobleaching, yielding about 103104 measuredphotons per fluorophore [23,24]. Background emissionfrom samples can easily overwhelm weak emissionsignals.

In a recent report, an approach is described that shouldprovide a readily measurable change in fluorescenceintensity in DNA hybridization formats [25]. Thisapproach increases the intensity relative to the back-ground and increases the number of detected photonsper fluorophore molecule by a factor of 10-fold or more.Figure 2a shows the sequence of the oligomers used inthis study. The thiolated oligonucleotide single-stranded(ss) DNA-SH was used as the capture sequence, whichbound spontaneously to the silver particles. The samplecontaining the silver-bound DNA was then positioned ina fluorometer followed by the addition of ss fluorescein-labeled DNA (Fl-DNA), in an amount approximatelyequal to the amount of silver-bound capture DNA(Figure 2b).

The fluorescence intensity began to increase immedi-ately upon mixing, and leveled off after about 20 min.This increase in intensity resulted from the localization ofss Fl-DNA near to the silver particles by hybridizationwith the capture DNA (Figure 2c). In the reported controlexperiments, ss DNA-SH was hybridized with ss Fl-DNAbefore deposition on silver particles; a similar 12-foldincrease in intensity upon immobilization on silver, ascompared with an equivalent amount of double-strandedFl-DNA-SH in solution, was observed [25].Current Opinion in Biotechnology 2005, 16:5562

58 Analytical biotechnology

Figure 2

ss DNA-SH

ss FI-DNA

Quartz

ss DNA-SH

ss FI-DNA

ds FI-DNA-SH

5-TCC ACA CAC CAC TGG CCA TCT TC-3-SH

5-TCC ACA CAC CAC TGG CCA TCT TC-3-SH

FI-3-AGG TGT GTG GTG ACC GGT AGA AG-5

FI-3-AGG TGT GTG GTG ACC GGT AGA AG-5

(a)

(b)

(c)50

40

30

20

10

00 5 10 15 20

Time (min)

Flu

ores

cenc

e in

tens

ity a

t 520

nm

ds FI-DNA-SHon SIFs afterhybridization

ss FI-DNAbefore hybridization

The application of MEF to DNA hybridization. (a) Structures of theDNA oligomers: ss DNA-SH (single-stranded thiolated oligonucleotide);

ss Fl-DNA (single-stranded fluorescent-labeled oligonucleotide).

(b) Schematic of the DNA oligomers bound to silver particles.

(c) The time-dependent hybridization of ss Fl-DNA to ss DNA-SH.

(Figure adapted from [25] with permission.)

Figure 3

6.0 6.5 7.0 7.5 8.0 8.5 9.0

1.0

1.5

2.0

Free in solutionImmobilized on MEF substrate

SNAFL-2-DextranExc 488 nm

pH

Inte

nsity

rat

io (

I >59

0/I 5

55/4

0)

The pH sensitivity of SNAFL-2 is retained in the presence of silver

nanoparticles. The intensity on the MEF substrate is about 30 to 40

times brighter than the intensity of probe immobilized on glass slides;

however, the ratios are similar. (Figure adapted from [26] with

permission.)Enhanced ratiometric fluorescence sensing

Enhanced ratiometric pH sensing using the pH-sensitivefluorophore seminaphthofluorescein carboxy SNAFL-2and SiFs has also been recently reported [26]. Metallicsurfaces can provide up to a 40-fold increase in probefluorescence intensity as compared to non-metallic sur-Current Opinion in Biotechnology 2005, 16:5562faces with the same probe coverage [26]. However,although the signal-to-noise ratio is significantly betterfor pH sensing, the emission wavelength ratiometricvalues are similar to those obtained in solution (i.e. theratios are the same) owing to the fact that the emissionfrom both the acidic and basic forms of the probe areenhanced to similar extents (Figure 3). This probably alsoreflects similar unmodified quantum yields of both forms.However, the substantial drop in probe lifetime precludesits utilization as a ratiometric lifetime sensor (see Equa-tions (3) and (4)). Hence, it is fair to conclude thatratiometric lifetime sensing will not be favorable by thisapproach.

Metal-enhanced multiphoton excitation

For multiphoton excitation of fluorescence, most of theexcitation occurs at the focal point of the excitation wherethe local intensity is the highest. For a two-photonabsorption process, the rate of excitation is proportionalto the square of the incident intensity. This suggests thattwo-photon excitation could be enhanced by a factor of3.8 108 [2,4]. Such an enhancement in the excitationrate is thought to provide selective excitation of fluoro-phores near to metal islands or colloids, even if thesolution contains a considerable concentration of otherfluorophores that could undergo two-photon excitation atthe same wavelength, but which are more distal from themetal surface.

Recently, the enhanced and localized multiphoton exci-tation of Rhodamine B [27], Pacific Blue, Lissamine andTexas Red [28] fluorescence near metallic silver islandshas been reported. A significant increase (up to 235-fold)in fluorescence emission intensity was seen for thesewww.sciencedirect.com

Metal-enhanced fluorescence: an emerging tool in biotechnology Aslan et al. 59fluorophores adjacent to metallic silver islands, accom-panied by a reduction in lifetime as compared to thatobserved using one-photon excitation [27,28]. Given thenow widespread use of multiphoton excitation in micro-scopy and medical imaging, this recent finding suggeststhe use of metallic nanostructures to both enhance andlocalize fluorescence.

Metal-enhanced planar immunoassays

MEF has also been utilized in the development of anenhanced detection limit sandwich-format immunoassayfor the cardiac marker myoglobin. In this importantexample of MEF, myoglobin was first captured on SiFsand glass surfaces coated with anti-myoglobin antibodies;the surfaces were then incubated with fluorophore-Figure 4

Wavelength (nm)

550 600 650 700 7500

1

2

3

4

5

40

80

Flu

ores

cenc

e (a

u)

Flu

ores

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u)

(a)

(b) (c)

Metal-enhanced planar immunoassays. (a) Schematic of a myoglobin-enhan

of the Rhodamine Red-X-labeled anti-myoglobin antibody bound to the surf

100 ng/mL on SiFs and on glass. (c) Fluorescence emission of Rhodamine

concentrations on SiFs (excitation 532 nm).

www.sciencedirect.comlabeled anti-myoglobin antibodies (Figure 4a). A 1015-fold increase in fluorescence emission was observedfrom the fluorophore-labeled antibody on the SiFs com-pared with that on the glass substrate alone (Figure 4b).Figure 4c shows that the detection range of myoglobin isbetween 10 and 1000 ng/mL. These results show that it ispossible to detect myoglobin concentrations below 50 ng/mL with an immunoassay performed using SiFs, muchlower than the clinical cut-off concentrations for myoglo-bin in healthy patients (100 ng/mL).

Metal-enhanced fluorescent probes

Fluorophores are essential in fluorescence-based technol-ogy and especially in DNA-based technologies. A recentreport studied the effects of SiFs on the emission spectral[Myoglobin] (ng/mL)

Rhodamine RedX-labeledanti-myoglobinantibody

Captureanti-myoglobinantibody

SIF on glasssubstrate

Blockingagent (BSA)

Myoglobin

10 100 10000

0

0

Current Opinion in Biotechnology

ced sandwich immunoassay on SiFs. (b) Fluorescence spectra

ace-immobilized myoglobin; the concentration of myoglobin was

Red-X-labeled anti-myoglobin antibody at different myoglobin

Current Opinion in Biotechnology 2005, 16:5562

60 Analytical biotechnology

Figure 5

Metal-enhanced fluorescent probes. (a) Emission spectra of Cy3-DNA and Cy5-DNA between quartz plates, with and without SiFs. (b,c)

Photographs of the corresponding fluorophores. Note that the photographs are taken through emission filters and, importantly, the increase

in emission intensity is not due to an increased excitation scatter from the silvered plates. Abbreviation: APS, aminopropyl silane. (Figure

adapted from [17] with permission.)properties of fluorophore-labeled DNA [17]. The emis-sion spectra of Cy3-DNA and Cy5-DNA on glass and onSiFs are shown in Figure 5a. The emission intensity isincreased two- to threefold on SiFs as compared withquartz slides (the control sample). The slightly largerincrease in emission intensity for Cy5-DNA is consistentwith previous observations of larger enhancements forlow quantum yield fluorophores [17]. Photographs of thelabeled oligomers on quartz and SiFs are also shown(Figures 5b,c). The emission from the labeled DNA onquartz is almost invisible, but is brightly visible on theSiFs. This difference in intensity results from an increasein the radiative decay rate of the fluorophore, which inturn results in an increase in the quantum yield (intensity)of the fluorophores [17].

Reports of the effects of silver particles on metalligandcomplexes, which are deemed to be useful as lumin-escence probes in biochemistry owing to their long lumin-escent decay times, have been reported [29]. Theemission spectral properties of [Ru(bpy)3]

2+ on SiFsand glass yielded several fold higher intensities on SiFs,accompanied by shortened lifetimes. These results areconsistent with an approximate 20-fold increase in radia-tive decay rate of the [Ru(bpy)3]

2+ in close proximity tosilver particles. It is likely, therefore, that silver particlesmight find use in increasing the detectability of emissionsfrom other metalligand complexes.Current Opinion in Biotechnology 2005, 16:5562Metal-enhanced solution assays

Nearly all of the work pertaining to MEF to date, hasdealt with fluorophores in close proximity to planar sur-faces. However, it might also be desirable to use metals ascolloidal suspensions for medical imaging, as they arepotentially injectable and silver and gold colloids arealready widely used in medicine. For example, suchapproaches are used in retinal angiography and couldbenefit from an enhanced indocyanine green and fluor-escein quantum yield and from increased photostability(see the article by Malicka et al. [16] and referencestherein).

To illustrate the usefulness of MEF in solution-basedbiosensing applications we acknowledge two recentapproaches. In the first approach, SiO2-coated silver col-loids provide for a solution-based enhanced-fluorescencesensing platform with a three- to fivefold enhancementtypically observed [30]. In the second approach,oligonucleotide-modified silver particles and afluorophore-labeled complementary oligonucleotidewere employed (Figure 6) [31]. An increased emissionfrom the fluorophore-labeled complementary oligonu-cleotide after hybridization in the presence of silverparticles has been observed. This result suggests apossible approach to DNA detection based on the aggre-gation of metallic nanoparticles bound by fluorophore-labeled oligonucleotides [31].www.sciencedirect.com

Metal-enhanced fluorescence: an emerging tool in biotechnology Aslan et al. 61

Figure 6

NaBH4

Displacement

COO

COOCOO

COO

COO

COO

COO

COO

COO

COO

COO

COO

COO

OOC

OOC

OOC

OOC

OOC

OOC

OOC

Hybridization

AgNO3 + Tiopronin Ag-S-Tiopronin

Metal-enhanced solution assays. One approach to the use of MEF in solution-based assays is to use oligonucleotide-modified silver particles

in solution together with fluorophore-labeled complementary oligonucleotides. The schematic illustrates the preparation, displacement by

thiolate oligonucleotides, and hybridization with fluorescein-labeled complementary oligonucleotides. The silver nanoparticles were protected

with a tiopronin monolayer. Silver particles are shown as gray spheres and fluorophore molecules as black ovals. (Figure adapted from [31]

with permission.)Interpretation of metal-enhancedfluorescence in terms of a radiatingplasmon modelOver the past five years, the exciting possibilities thatmetalfluorophore interactions potentially have to offer tomedical imaging and diagnostics have been realized.Although most of these approaches have been poisedfrom a sensing perspective, and therefore phenomenolo-gical in nature, the descriptive nature of the underlyingphysics has been traditionally centered on excited distalfluorophores in resonance with surface plasmon electrons.That is, the fluorophore is excited directly and sponta-neously emits; the spectral changes observed then resultfrom the excited-state resonance interaction with the freeelectrons on the metallic surface.

More recently, however, the interpretation of the sameresults has shifted somewhat to a model whereby non-radiative energy transfer occurs from distal excited fluor-ophores to the surface plasmon electrons. The surfaceplasmon electrons, in turn, radiate (under certain condi-tions) the photophysical characteristics of the couplingfluorophore. This new model has been referred to as theradiating plasmon model [32].

The extinction properties of metal particles can beexpressed by a combination of both absorption (CA)and scattering (CS) factors, when the particles are sphe-www.sciencedirect.comrical and have sizes comparable to the incident wave-length of light:

CE CA CS k1Ima k416p

jaj2 (5)

where k1 = 2pn1/l0 and is the wave vector of the incidentlight in medium 1 and a is the polarizability of the spherewith radius r and is given by:

a 4pr3em e1=em 2e1 (6)

where em is the complex dielectric constant of the metal.The first term represents the cross-section owing toabsorption (CA) and the second term the cross-sectionowing to scattering (CS). Current interpretation of MEF istherefore underpinned by the scattering component ofthe metal extinction (i.e. the ability of fluorophore-coupled plasmons to radiate [32]).

ConclusionsWe have reviewed recent fluorophoremetal architecturesthat offer enhanced spectral properties of the fluorophores,such as increased quantum yields, increased rates of exci-tation and energy transfer, and enhanced fluorophorephotostability (reduced lifetimes). Metallic silver can bereadily deposited by a variety of methods, on demand,using a range of approaches and in biologically inert envir-onments. In our opinion these fluorophoremetal effectsCurrent Opinion in Biotechnology 2005, 16:5562

62 Analytical biotechnologyoffer unique perspectives in clinical chemistry and bio-chemistry, providing for improved background suppres-sion, increased detection limits and even localizedexcitation. MEF is an emerging technology that in severalyears is likely to be widespread throughout fluorescence-based applications of biotechnology.

AcknowledgementsThe authors would like to thank the National Institutes of Health forfinancial support (National Center for Research Resources, RR-08119and R21GM070929), Philip Morris USA Inc. and Philip MorrisInternational, and the National Institute for Biomedical Imaging andBioengineering (EB-1690). The authors also wish to thank the MedicalBiotechnology Center, University of Maryland Biotechnology Institute forpartial salary support to JRL, CDG and IG. Others involved in this workinclude Zygmunt Gryczynski, Joanna Lukomska, Jian Zhang,Ramachandram Badugu, Slawomir Makowiec, Kazimierz Nowacyzk,Meng Wu and Jun Huang.

References and recommended reading

of special interest of outstanding interest

1. Lakowicz JR: Radiative decay engineering: biophysical andbiomedical applications. Anal Biochem 2001, 298:1-24.

2. Lakowicz JR, Shen Y, DAuria S, Malicka J, Fang J, Gryczynski Z,Gryczynski I: Radiative decay engineering 2. Effects of silverisland films on fluorescence intensity, lifetimes, andresonance energy transfer. Anal Biochem 2002, 301:261-277.

3. Lakowicz JR, Shen Y, Gryczynski Z, DAuria S, Gryczynski I:Intrinsic fluorescence from DNA can be enhanced by metallicparticles. Biochem Biophys Res Com 2001, 286:875-879.

4. Gryczynski I, Malicka J, Shen Y, Gryczynski Z, Lakowicz JR:Multiphoton excitation of fluorescence near metallic particles:enhanced and localized excitation. J Phys Chem B 2002,106:2191-2195.

5. Geddes CD, Lakowicz JR: Metal-enhanced fluorescence. JFluores 2002, 12:121-129.

6. Gersten J, Nitzan A: Spectroscopic properties of moleculesinteracting with small dielectric particles. J Chem Phys 1981,75:1139-1152.

7. Weitz DA, Garoff S, Gersten JI, Nitzan A: The enhancement ofRaman scattering, resonance Raman scattering andfluorescence from molecules absorbed on a rough silversurface. J Chem Phys 1983, 78:5324-5338.

8. Sokolov K, Chamanov G, Cotton TM: Enhancement of molecularfluorescence near the surface of colloidal metal films. AnalChem 1998, 70:3898-3905.

9. Drexhage KH: Interaction of light with monomolecular dyelasers. In Progress in Optics. Edited by E Wolfe. North-Holland:Amsterdam; 1974:161-232.

10. Geddes CD, Cao H, Gryczynski I, Gryczynski Z, Fang J, LakowiczJR: Metal-enhanced fluorescence due to silver colloids on aplanar surface: potential applications of indocyanine green toin vivo imaging. J Phys Chem A 2003, 107:3443-3449.

11. Aslan K, Leonenko Z, Lakowicz JR, Geddes CD: Fast and slowdeposition of silver nanorods on to glass substrates. J PhysChem B: in press.

12. Geddes CD, Parfenov A, Lakowicz JR: Photodeposition of silvercan result in metal-enhanced fluorescence. Appl Spectrosc2003, 57:526-531.

13. Geddes CD, Parfenov A, Roll D, Fang J, Lakowicz JR:Electrochemical and laser deposition of silver for use in metal-enhanced fluorescence. Langmuir 2003, 19:6236-6241.Current Opinion in Biotechnology 2005, 16:556214. Forster Th: Intermolecular energy migration and fluorescence.Ann Phys 1948, 2:55-75.

15.

Lakowicz JR, Malicka J, DAuria S, Gryczynski I: Release of theself-quenching of fluorescence near silver metallic surface.Anal Biochem 2003, 320:13-20.

First report of the release of self-quenching of fluorophores near-tometallic surfaces.

16. Malicka J, Gryczynski I, Geddes CD, Lakowicz JR: Metal-enhanced emission from indocyanine green: a new approachto in vivo imaging. J Biomed Opt 2003, 8:472-478.

17. Lakowicz JR, Malicka J, Gryczynski I: Silver particles enhanceemission of fluorescent DNA oligomers. Biotechniques 2003,34:62-68.

18. Brown PO, Botstein D: Exploring the new world ofthe genome with DNA microarrays. Nat Genet Supp 1999,21:33-37.

19. Schena M, Heller RA, Theriault TP, Konrad K, Lachenmeier E,Davis RW: Microarrays: biotechnologys discovery platformfor functional genomics. Trends Biotechnol 1998,16:301-306.

20. Komurian-Pradel F, Paranhos-Bacala G, Sodoyer M, Chevallier P,Mandrand B, Lotteau V, Andre P: Quantitation of HCV RNA usingreal-time PCR and fluorimetry. J Virol Methods 2001,95:111-119.

21. Walker NJ: A technique whose time has come. Science 2002,296:557-559.

22. Difilippantonio MJ, Ried T: Technicolor genome analysis. InTopics in Fluorescence Spectroscopy, DNA Technology, Vol 7.Edited by JR Lakowicz. Kluwer Academic Publishers/PlenumPress: New York; 2003: 291-316.

23. Soper SA, Nutter HL, Keller RA, Davis LM, Shera EB: Thephotophysical constants of several fluorescent dyespertaining to ultrasensitive fluorescence spectroscopy.Photochem Photobiol 1993, 57:972-977.

24. Ambrose WP, Goodwin PM, Jett JH, VanOrden A, Werner JH,Keller RA: Single molecule fluorescence spectroscopy atambient temperature. Chem Rev 1999, 99:2929-2956.

25.

Malicka J, Gryczynski I, Lakowicz JR: DNA hybridization assaysusing metal-enhanced fluorescence. Biochem Biophys ResCommun 2003, 306:213-218.

First report of MEF to follow DNA hybridization.

26. Aslan K, Lakowicz JR, Geddes CD: Enhanced ratiometric pHsensing using SNAFL-2 on silver island films: metal-enhancedfluorescence sensing. J Fluores, 2005: in press.

27. Lakowicz JR, Gryczynski I, Malicka J, Gryczynski Z, Geddes CD:Enhanced and localized multiphoton excited fluorescencenear metallic silver islands: metallic islands can increaseprobe photostability. J Fluores 2002, 12:299-302.

28. Maliwal BP, Malicka J, Gryczynski I, Gryczynski Z, Lakowicz JR:Fluorescence properties of labeled proteins near silver colloidsurfaces. Biopolymers 2003, 70:585-594.

29. Gryczynski I, Malicka J, Holder E, DiCesare, Lakowicz JR: Effectsof metallic silver particles on the emission properties of[Ru(bpy)3]

2+. Chem Phys Lett 2003, 372:409-414.

30.

Aslan K, Lakowicz JR, Szmacinski H, Geddes CD: Metal-enhanced fluorescence solution-based sensing platform.J Fluores 2004, 14:677-679.

First report of MEF in solution with colloids that result in enhancedintensities upon aggregation.

31. Zhang J, Malicka J, Gryczynski I, Lakowicz JR: Oligonucleotide-displaced organic monolayer-protected silver nanoparticlesand enhanced luminescence of their salted aggregates.Anal Biochem 2004, 330:81-86.

32. Lakowicz JR: Radiative decay engineering 5. Non-quenchingmetallic surfaces and plasmon emission. Anal Biochem 2005:in press.www.sciencedirect.com

Metal-enhanced fluorescence: an emerging tool in biotechnologyIntroductionMethodologies for the preparation of silver nanoparticlesApplications of metal-enhanced fluorescence to biotechnologyUltrabright over-labeled proteins: a new class of probes based on MEFDNA hybridizationEnhanced ratiometric fluorescence sensingMetal-enhanced multiphoton excitationMetal-enhanced planar immunoassaysMetal-enhanced fluorescent probesMetal-enhanced solution assays

Interpretation of metal-enhanced fluorescence in terms of a radiating plasmon modelConclusionsAcknowledgementsReferences and recommended reading